The long term goal of this proposal is to establish the molecular basis by which eukaryotic polytopic membrane proteins integrate, fold and assemble in the lipid bilayer of the endoplasmic reticulum (ER). Aquaporins represent a prototype class of polytopic proteins that contain six transmembrane (TM) segments and form selective water-permeable channels in cell membranes. At least six aquaporins are expressed in the mammalian kidney where they play critical roles in fluId and electrolyte homeostasis. While the basic steps of aquaporin assembly in ER have recently been described, very little is known about how cellular machinery mediates specific translocation, membrane integration, and folding events required to establish AQP topology. This is a major limitation in our understanding of normal renal physiology and in particular, pathologic states where aquaporin folding is disrupted, e.g. nephrogenic diabetes insipidus. Recent studies from our laboratory, now provide for the first time, a means to define the molecular interactions responsible for different and novel polytopic protein folding pathways. Because of their significant role in normal physiology, their relatively simple architecture, and their unusual biogenesis mechanisms, aquaporins represent ideal candidates for such a study. The specific aims are: i) to characterize different molecular pathways of aquaporm assembly in the ER membrane, ii) to define how primary structural determinants generate variations in these folding pathways, and iii) to identify novel components within the ER that are required for specialized aspects of aquaporin biogenesis. Proposed experiments will use cell free translation systems to incorporate photoactive crosslinking probes at engineered sites in native, mutant and chimeric aquaporin proteins. These experiments will define molecular interactions between the nascent polypeptide and ER translocation machinery that mediate protein folding and determine how subtle variations in sequence influence normal biogenesis events and topologic outcome. Finally novel factors required for aquaporin maturation will be identified by fractionation and heterologous reconstitution of translocation competent ER membranes that exhibit distinct differences in aquaponn maturation. Together these studies will provide a major advance in our knowledge of the molecular events involved in polytopic protein biogenesis and will establish a foundation for understanding how inherited mutations disrupt biogenesis in human disease.